DESCRIPTION: The overall aim of the proposal is to elucidate the multiple functions of the NS3 and NS5 proteins of dengue virus type 2 in viral RNA replication and 5' genomic RNA capping. A clear understanding of viral RNA replication is important to understand the mechanism of viral pathogenesis. NS3 in association with the NS2B protein is a serine protease required for the processing of the viral polyprotein. NS3 also has the conserved motifs of a NTPase/RNA helicase, and it also has a 5' RNA triphosphatase activity, required for viral replication and capping respectively. NS5 has motifs for RNA methyltransferase and RNA-dependent RNA polymerase. In vitro RNA synthesis will be sought for using isolated membrane bound replication complexes that are specifically dependent on exogenously added viral RNA templates containing the 3' terminus of the genomic RNA. Viral proteins and template sequences required for the minus strand synthesis will be identified by mutagenesis. The RNA helicase of NS3 and the RdRp activity of NS5 involved in replication will be characterized. In addition, the viral proteins and their enzyme activities required for 5' RNA capping will be identified. Since these enzyme activities are uniquely required for the virus lifecycle, they are potential targets for antiviral strategies.